Little is understood about the mechanisms coordinating the balance between epithelial cell proliferation and cell differentiation necessary to maintain ocular surface health. In the past grant period, this project focused on the role of Matrix Metalloproteinase (MMP) gelatinase B (gelB; MMP-9), expression of which is induced at the front of the corneal epithelium migrating to resurface a wound. The novel finding was made that gelB modulates the proliferative balance in the regenerating epithelium. GelB deficiency in knock out mice accelerated cell proliferation in the regenerating epithelium, associated with premature accumulation of the cytokine IL-1alpha. In contrast, it was learned that this does not work in reverse, that IL-1 does not stimulate gelB expression in the absence of any other input. This is different from its effect on expression of other MMPs, and needs to be further understood. GelB deficiency was also associated with a delay in activation of Smad2 transcription factor in the regenerating epithelium, suggesting the mechanism for enhancing the proliferative rate. Another novel finding was that the transcription factor Pax-6 controls the GelB promoter in both a positive and negative manner, and is induced along with gelB at the migrating front of the corneal epithelium. Preliminary data suggests that Pax-6 deficiency is similar to gelB deficiency in its effect on ocular resurfacing. This new proposal builds on the conceptual framework developed in the last grant period, extending the studies to answer some of the questions raised by the new findings. A new focus will be signaling through the Epidermal Growth Factor Receptor (EGFR), and its modulation by G protein-coupled receptors (GPCRs), which are activated by corneal nerves. The: following questions will be addressed: 1) What controls gelB expression in the wound environment and can corneal nerves alter the capacity of IL-1 to control gelB expression? 2) How does gelB control IL-lalpha expression, and does IL-1alpha affect epithelial migration or proliferation? If so, can this explain the gelB-deficient phenotype? 3) How does gelB control Smad2 activity and how do nerves interact? 4) What are the larger effects of Pax-6 on maintenance of the ocular surface? Results of these planned experiments will provide basic information about the biology of the ocular surface, and will identify molecular targets for drug development and gene therapy to correct ocular surface disorders. They will be further useful in solving problems associated with bioengineering of artificial corneas.